Curated Optogenetic Publication Database

Abstract: Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

Abstract: Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

Abstract: Cellular functioning relies on active transport of organelles by molecular motors. To explore how intracellular organelle distributions affect cellular functions, several optogenetic approaches enable organelle repositioning through light-inducible recruitment of motors to specific organelles. Nonetheless, robust application of these methods in cellular populations without side effects has remained challenging. Here, we introduce an improved toolbox for optogenetic control of intracellular transport that optimizes cellular responsiveness and limits adverse effects. To improve dynamic range, we employed improved optogenetic heterodimerization modules and engineered a photosensitive kinesin-3, which is activated upon blue light-sensitive homodimerization. This opto-kinesin prevented motor activation before experimental onset, limited dark-state activation, and improved responsiveness. In addition, we adopted moss kinesin-14 for efficient retrograde transport with minimal adverse effects on endogenous transport. Using this optimized toolbox, we demonstrate robust reversible repositioning of (endogenously tagged) organelles within cellular populations. More robust control over organelle motility will aid in dissecting spatial cell biology and transport-related diseases.

Abstract: Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.